The present invention relates to electrospray ionization (ESI) devices or other ion injection spray devices for use in LC/MS. LC/MS is an important tool in the analysis of many chemical compounds including biomolecules which are important to human health and longevity. Specifically, LC/MS can be used to isolate, identify, characterize and quantify a wide range of sample molecules. The analysis of samples by LC/MS consists of four main steps; 1) LC separation of the different molecules in a sample, 2) formation and desolvation of sample molecule ions, 3) mass analysis to separate the ions from one another according to their mass to charge ratios, and 4) detection of the ions. A variety of means exist in the field of LC/MS to perform each of these functions. The particular combination of means used in a given LC/MS system determines the characteristics of that specific system.
To mass analyze ions, for example, one might use an ion trap analyzer, where ions are trapped by a radio frequency (RF) quadrupole field and mass selective ejected by scanning RF amplitude and/or dc voltage. Other mass analyzers include the quadrupole (Q), the ion cyclotron resonance (ICR), the sector (using a magnetic or electrostatic field or both), and the time of flight (TOF) analyzers.
Before mass analysis can begin, however, gas phase ions must be formed from the sample molecules. If the sample molecules are sufficiently volatile, ions may be formed by electron impact (EI) or chemical ionization (CI). For solid samples, ions can be formed by desorption/ionization of the sample molecules by bombardment with high energy particles. For liquid phase sample molecules, atmospheric pressure ionization (API) is currently the technique of choice. One of the more widely used API methods, known as electrospray ionization (ESI), was first suggested by Dole et al. (M. Dole, L. L. Mack, R. L. Hines, R. C. Mobley, L. D. Ferguson, M. B. Alice, J. Chem. Phys. 49, 2240, 1968). In ESI, analytes in solution and sprayed from a needle and the spray is induced by the application of a potential difference between the spray tip (where the liquid emerges) and a counter electrode. By subjecting the emerging liquid to a strong electric field, it becomes charged, and as a result, it “breaks up” into smaller particles if the charge imposed on the liquid's surface is strong enough to overcome the surface tension of the liquid (i.e., as the particles attempt to disperse the charge and return to a lower energy state). This results in the formation of fine, charged droplets of solution containing the analyte molecules. These droplets further evaporate leaving behind gas phase analyte ions.
Electrospray mass spectrometry (ESI-MS) was introduced by Yamashita and Fenn (M. Yamashita and M. B. Fenn, J. Phys. Chem. 88, 4671, 1984). To establish this combination of ESI and MS, ions had to be formed at atmospheric pressure, and then introduced into the vacuum system of a mass analyzer via a differentially pumped interface. The combination of ESI and MS afforded scientists the opportunity to mass analyze a wide range of samples, and ESI-MS is now widely used in the analysis of biomolecules and other complex organic molecules.
Over the past two decades, a number of means and methods of electrospray useful to LC/MS have been developed. For higher LC flow rates (i.e. 50-5000 ul/min), pneumatic assisted electrospray has become the technique of choice (A. P. Bruins, T. R. Covey, and J. D. Henion, Anal. Chem., 59, 2642, 1987, and Henion et al, U.S. Pat. No. 4,861,988). This technique uses a gas flowing past the ESI spray tip to assist in the formation and desolvation of charged droplets. Although the gas assists in the formation of the spray and makes the operation of the electrospray ionization (ESI) easier and more robust, the excess gas dilutes the sample ions, resulting in lower ion transfer efficiency and a loss of sensitivity.
For lower flow LC/MS (10-1000 nl/min), nanospray ionization (NSI) has become the technique of choice (M. S. Wilm and M. Mann, Int. J. Mass Spectrom. Ion Processes, 136-167, 1994; and M. Mann and M. S. Wilm, U.S. Pat. No. 5,504,329). U.S. Pat. No. 5,504,329 is incorporated herein by reference in its entirety, with various details of NSI being utilized with the apparatus and method of this invention. NSI utilizes very low liquid flows and a very narrow spray tip outlet placed very close to the MS inlet, which results in the formation of very small spray droplets which can be desolvated without gas assistance. Although the ion signal provided by NSI in conjunction with MS is essentially the same as with conventional ESI, MS is a concentration sensitive detection technique which makes NSI the best technique for high sensitivity applications. Since no gas is used in NSI, high ion transfer efficiency can be achieved, but at a cost of ease of use and robustness relative to pneumatic assisted electrospray.
When using NSI-MS, the liquid flow rate, solvent composition, spray tip voltage, spray tip design, spray tip integrity and the position of the spray tip outlet relative to the MS inlet are all critical for good spray stability which results in proper ionization, desolvation and ion transfer efficiency. NSI spray tips are generally made by pulling and cutting fused silica tubing to make the very small ID/OD tips required for stable spray at nanoliter per minute flow rates, but these tips are difficult to reproduce, fragile to handle and easy to clog. Because of these limitations, NSI can be difficult to set up and maintain, making it poorly suited for analyses requiring robust operation.
Since NSI is generally limited to flow rates below 1 μl/min, samples must be separated using nanoLC which has its own share of problems and limitations. NanoLC requires specialized instrumentation and careful attention to details to insure optimal performance. NanoLC columns (<150 um ID) have limited sample capacity, require specialized sample injection protocols to load large sample volumes and lack the robustness of larger LC columns. Finally, the low flow rates used in nanoLC/NSI-MS typically result in longer sample analysis time, making this technique poorly suited to high throughput applications like biomarker validation and pharmaceutical development.
Several attempts have been made to develop commercially viable microspray ionization (MSI) sources in an effort to overcome the limitations imposed by NSI, but these MSI sources have not been very well accepted. Although these MSI sources, which are basically miniaturized versions of pneumatic assisted ESI, do offer increased stability and work at higher LC flow rates versus NSI, the added gas flow still results in a lower ion transfer efficiency and a unacceptable loss in sensitivity for most researchers.
The applicants have recognized the need for a LC/MS electrospray apparatus and method that can overcome the limitations imposed by ESI, MSI and NSI, without compromising the ion transfer efficiency critical to high sensitivity applications. This apparatus and method provide simple, robust operation over a wide dynamic flow range and maintain high ion transfer efficiency independent of the LC flow rate. This apparatus and method can be be simple to set up and use (“Plug and Play”), operate continually with minimal maintenance and provide both high sensitivity and high throughput operation, especially at flows from 0.1-100 ul/min.